PHLPP1/Akt/Mst1信号通路调控的细胞凋亡和自噬在缺血后处理对糖尿病心肌保护失敏感中的作用及机制研究

Ischemic postconditioning (IPostC) protects the heart from ischemia reperfusion (IR) injury. However, IPostC cardioprotection is lost in diabetic hearts and its mechanisms remain unclear. PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) suppresses protein kinase B (Akt) and in turn activates Ste20-like protein kinase (Mst1) to promote apoptosis and inhibit autophagy. Our pilot experiments revealed that IPostC exerted cardioprotection against myocardial IR injury via decreasing the expression of PHLPP1 to augment Akt activity and inhibit Mst1 activity to reduce apoptosis and increase autophagy; however, these effects were not present in the myocardium of diabetic mice. Our data also revealed that diabetes was concomitant with cardiac overexpression of PHLPP1, elevation of Mst1 activity, and reduction of Akt activity and that downregulation of PHLPP1 expression alleviates the hypoxia/reoxygenation-induced injury in H9c2 cells exposed to high glucose. Hence, we speculate that PHLPP1 signaling plays an important role in the IPostC-induced cardioprotection via modulating apoptosis and autophagy of myocardial cells and that the aberrant expression of PHLPP1 to impair the protective signaling is possibly the key role resulting in myocardial desensitization to IPostC in diabetic rodents. This project intends to employ multiple in vivo and in vitro experimental approaches to examine the aforementioned hypotheses. The resulting data may provide a mechanistic basis for the clinical application of IPostC in ischemic heart disease.

缺血后处理(IPostC)对心肌缺血再灌注损伤具有保护作用，但是保护作用在糖尿病中消失，机制仍不清楚。PH结构域富含亮氨酸重复序列蛋白激酶1(PHLPP1)可灭活蛋白激酶B(Akt)而激活哺乳动物STE-20样激酶1(Mst1)，进而促进凋亡和抑制自噬。预实验发现IPostC降低PHLPP1的表达而提升Akt的活性和抑制Mst1的活性，减少细胞凋亡和增加细胞自噬，最终减轻心肌损伤，而以上作用在糖尿病中消失。预实验同时发现在糖尿病心肌中PHLPP1过度表达，导致Akt活性显著降低和Mst1活性增加，而通过下调PHLPP1的表达可减轻缺氧再复氧后高糖培养细胞的损伤。由此推测PHLPP1表达异常导致保护通路调节受损是IPostC对糖尿病心肌保护失敏感的关键。本课题拟采用多种实验技术，在整体和细胞水平阐明以上推论，为IPostC更好的应用于临床提供理论依据。

背景 缺血后处理（IPostC）可减轻心肌缺血/再灌注损伤，但在糖尿病中保护作用消失。PH结构域富含亮氨酸的重复蛋白磷酸酶1(PHLPP1)是一种丝氨酸/苏氨酸磷酸酶，与心血管疾病和心肌缺血再灌注损伤等多种病理过程有关，可失活Akt激酶或激活Mst1激酶。我们的前期研究发现，PHLPP1在糖尿病心脏中的表达上调。我们推测糖尿病组织中PHLPP1过表达与IPostC在糖尿病心肌中的保护作用消失有关。.方法 健康或糖尿病小鼠接受45分钟冠状动脉闭塞，随后再灌注2小时，部分组接受缺血后处理。在细胞实验中，H9c2细胞培养于正常葡萄糖或者高糖培养基中，部分组接受4小时的缺氧，随后进行4小时的复氧培养。检测指标包括心肌梗死面积、心肌酶释放、细胞凋亡和氧化应激。通过蛋白印迹方法测量目标蛋白及其磷酸化的表达水平。通过CCK-8试剂盒测量H9c2细胞的活力。利用JC-1染色法检测心肌细胞中的线粒体膜电位。.结果 IPostC显着降低非糖尿病小鼠的缺血后心肌梗死、细胞凋亡、血浆肌酸激酶-MB的释放和氧化应激水平，同时增加p-Akt及降低PHLPP1和p-Mst1的表达，但在糖尿病小鼠中以上作用均消失。敲低PHLPP1表达或Mst1抑制剂XMU-MP-1减轻缺氧/复氧 (HR)导致的H9c2细胞的损伤，但相关作用被PI3K/Akt抑制剂LY294002消除。缺氧后处理（HPostC）显著减轻HR诱导的心肌细胞损伤和氧化应激，同时增加增加p-Akt及降低PHLPP1和p-Mst1的表达，但以上作用在高糖培养的H9c2细胞中丧失。此外，HPostC结合敲低PHLPP1表达或单独敲低PHLPP1表达均可减少培养于高糖的H9c2细胞的死亡和氧化应激，而以上作用被LY294002消除。.总结 IPostC通过PHLPP1/Akt/Mst1信号通路减轻心肌缺血再灌注损伤，而该通路的表达异常可能是IPostC在糖尿病心肌中保护功能缺失的原因。